Calcium signaling pathways form a network that is critical for normal operations of nearly all cell types in the human body and in nearly all eukaryotic species. Drugs that block different components of the network are used to therapeutically to treat many kinds of disorders. Drugs that specifically block calcineurin, a Ca2+- dependent protein phosphatase, are potent immunosuppressants used widely to prevent immunological rejection of transplanted organs. The major goal of this proposal is to increase our understanding of calcineurin regulation and functioning within the calcium signaling network. We discovered a novel family of proteins conserved from yeast to humans that directly bind and regulate calcineurin. Through genetic studies in yeast, we proposed a novel hypothesis where these regulators of calcineurin (RCNs) cycle between stimulatory and inhibitory states based on phosphorylation of a conserved linker domain in the proteins. Here we plan to critically evaluate this hypothesis in a careful series of biochemical and genetic experiments using human DSCR1/MCIP1, yeast Rcn1p, and yeast Rcn2p which we recently discovered as a highly divergent member of the RCN family. In yeast, calcineurin inhibits a plasma membrane high-affinity Ca2+ channel and a vacuolar H+/Ca2+ exchanger, which help to control cytosolic Ca2+ and the activities of Ca2+-dependent enzymes such as calcineurin. We have identified several new subunits or regulators of these enzymes and we plan to determine the molecular interactions among these proteins, which of the factors are direct substrates of calcineurin, and whether the effects of calcineurin are necessary and sufficient for regulation of the enzymes. Finally, we discovered the first subunit or regulator of a distinct low affinity Ca2+ channel in yeast and identified it as a member of the human claudin/stargazin superfamily of membrane proteins that are known to regulate ion fluxes in many tissues. We propose genetic experiments to identify and characterize additional components and regulators of this novel channel in yeast. Our findings will shed light on the structure and function of the calcium signaling network in eukaryotes, which may accelerate the development of improved therapeutics for organ transplantation, treatment of heart failure, and treatment of antibiotic-resistant fungal infections.